Our laboratory is interested in understanding how eukaryotic cells ensure the maintenance of telomeres, the natural ends of linear eukaryotic chromosomes. Evolutionarily conserved shelterin and CST (CTC1/Cdc13- STN1-TEN1) complexes play essential roles in telomerase recruitment and protection of telomeres against DNA repair and checkpoint factors. Stable maintenance of telomeres is critical to preserve genomic integrity and prevent accumulation of undesired mutations that might lead to tumor formation. Regulation of telomere structures and telomerase also affect cell proliferation and tissue maintenance in aging organisms. Therefore, basic mechanistic studies investigating how telomere and DNA damage response proteins collaborate in proper telomere maintenance should provide critical insights necessary to help devise more effective treatment strategies against tumors or other age related diseases. Our proposed research projects utilize fission yeast Schizosaccharomyces pombe. Fission yeast telomeres serve as a good model for human telomeres, since proteins involved in telomere maintenance are highly conserved between fission yeast and humans. In addition, the ability of fission yeast cells to survive severe telomer dysfunction by circularizing all their chromosomes provides unique opportunities to study functional contributions of essential telomere maintenance factors, without being hindered by cell lethality. During the past funding period, we identified phosphorylation of the shelterin subunit Ccq1 at Threonine-93 (Thr93) by DNA damage checkpoint kinases Rad3ATR and Tel1ATM as the critical post-translational modification that promotes interaction between Ccq1 and the telomerase regulatory subunit Est1 to allow telomerase recruitment. Furthermore, we demonstrated that fission yeast shelterin and Stn1-Ten1 complexes interact, and that SUMOylation of the shelterin subunit Tpz1TPP1 at Lysine-242 (Lys242) facilitates shelterin-Stn1-Ten1 interaction to allow efficient accumulation of Stn1-Ten1 at telomeres and to limit telomerase-dependent telomere elongation. Our detailed analyses of temporal binding patterns for DNA polymerases, telomerase, shelterin and Stn1 also demonstrated that Rap1, Poz1 and Stn1-Ten1 promote timely dissociation of telomerase from telomeres by promoting timely recruitment of Pol? to complete lagging strand synthesis at telomeres. We have also successfully identified numerous phosphorylation sites on Tpz1TPP1 and Ccq1 by mass spectrometry. For the upcoming funding period, we propose experiments to define the functional significance of additional phosphorylation sites in Tpz1 and Ccq1 (Aims1-2) and investigate how shelterin, Stn1-Ten1 and Pol? collaborate to ensure telomere maintenance (Aim3). Since TPP1 is also highly phosphorylated and CST- and shelterin- dependent coordination of lagging strand synthesis and telomerase recruitment also play critical roles in telomere maintenance in mammalian cells, successful completion of the proposed experiments will likely have major impacts on future mammalian telomere studies.